RESUMO
Hybridoma antibodies (Hab) were prepared against the Karp, Gilliam and Kato strains of Rickettsia tsutsugamushi and were examined for homologous and heterologous reactivity using an indirect immunofluorescence assay. Strain-specific Hab demonstrated homologous IFA titers ranging from 1/320 to 1/1,280 and did not react (less than 1/10) with the heterologous strains. The cross-reactive Hab generally reacted equally with all three strains in the scrub typhus group; however, there were some Hab that reacted with only one of the two heterologous strains tested. The Hab also were examined in enzyme-linked immunosorbent assays with scrub typhus antigens eluted from SDS-polyacrylamide gels. Most Hab reacted with either one or several of the six eluted antigens detected with a polyclonal immune serum. It was also observed that strain-specific and cross-reactive Hab sometimes reacted with the same antigen, suggesting the existence of multiple antigenic determinants in one electrophoretic peak. The data suggest that strain-specific Hab can be used in the indirect immunofluorescence assay to identify isolates of R. tsutsugamushi without the cross-reactions usually observed with polyclonal antisera, and that they are useful probes for detection and analysis of rickettsial antigens.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Bactérias/imunologia , Orientia tsutsugamushi/imunologia , Anticorpos Antibacterianos/imunologia , Reações Cruzadas , Epitopos , Especificidade da EspécieRESUMO
Somatic cell hybrids were obtained by fusion of canine peritoneal macrophages and SV40-transformed human skin fibroblasts. A cell line (WRH-2) was established from a single isolated hybrid clone. The WRH-2 cell line has been serially passaged 60 times and has a population doubling time of approximately 24 hours. Karyotypic analysis showed the modal number of chromosomes to be 80, with a selective segregation of canine chromosomes. Expression of incorporated canine DNA was substantiated by cellular enzyme activities and antigen expression. The susceptibility of 5% to 7% of WRH-2 cells to Ehrlichia canis infection was associated with phagocytic properties of these cells. Magnetic separation of phagocytic cells after ingestion of carbonyl iron resulted in a significant enhancement of the phagocytic population with a concomitant increase in the percentage of cells susceptible to ehrlichial infection. Serial passage of the selected subpopulation of hybird cells, however, resulted in a rapid diminution in the percentage of phagocytic cells.
Assuntos
Transformação Celular Viral , Ehrlichia/crescimento & desenvolvimento , Células Híbridas/citologia , Macrófagos/citologia , Rickettsiaceae/crescimento & desenvolvimento , Vírus 40 dos Símios/crescimento & desenvolvimento , Animais , Antígenos/análise , Líquido Ascítico/citologia , Fusão Celular , Linhagem Celular , Separação Celular , Cães , Fibroblastos/citologia , Fibroblastos/imunologia , Humanos , Células Híbridas/imunologia , Cariotipagem , Macrófagos/imunologia , Fagocitose , Pele/citologia , Especificidade da EspécieRESUMO
Canine peritoneal macrophages were obtained by repeated peritoneal lavage of dogs at 14 day intervals. Intraperitoneal administration of sterile mineral oil increased the leukocyte yield approximately 20-fold and the macrophage recovery approximately 35-fold. Cell recovery was maximum 7 to 21 days after administration of oil and declined slightly by 35 days. Restimulation with a 2nd injection of oil promptly revitalized cell recovery. Peritoneal macrophage cultures were well established by 6 days after seeding and were maintained for at least 30 days. Initially, cultured macrophages were mononuclear, but binuclear cells appeared after 6 days' cultivation and multinucleated giant cells were observed after 14 days. Canine peritoneal macrophages and peripheral blood monocytes were equally susceptible to infection with Ehrlichia canis. Infected cells were detected by 60 hours after inoculation, and replication was evident by 12 to 18 days.
Assuntos
Ehrlichia/crescimento & desenvolvimento , Macrófagos/microbiologia , Rickettsiaceae/crescimento & desenvolvimento , Animais , Líquido Ascítico/citologia , Células Cultivadas , Cães , Feminino , Técnicas In Vitro , Macrófagos/citologia , Monócitos/citologia , Monócitos/microbiologia , Irrigação TerapêuticaRESUMO
Antibodies to Rickettsia rickettsii were detected by indirect immunofluorescence in sera from 149 of 467 dogs (32%) examined from 4 military installations located in Kentucky, North Carolina, Pennsylvania, and Virginia. The prevalence at individual installations ranged from 4.3% at Fort Knox, Ky, to 63.4% at Fort Bragg, NC. Most of the seropositive dogs were in the working and sporting groups of dogs. The difference in antibody prevalence between sexes was not significant. Serologic responses were related to R rickettsii infection, although antibodies to R montana also were detected in a few of the sera. Comparison of serodiagnostic methods indicated that the indirect fluorescent antibody test was more sensitive than was the indirect hemagglutination test for obtaining survey data on the prevalence of Rocky Mountain spotted fever in the area.
Assuntos
Anticorpos Antibacterianos/análise , Doenças do Cão/epidemiologia , Rickettsia rickettsii/imunologia , Febre Maculosa das Montanhas Rochosas/veterinária , Animais , Doenças do Cão/diagnóstico , Doenças do Cão/imunologia , Cães , Feminino , Imunofluorescência/veterinária , Testes de Hemaglutinação/veterinária , Humanos , Kentucky , Masculino , North Carolina , Pennsylvania , Rickettsia/imunologia , Febre Maculosa das Montanhas Rochosas/diagnóstico , Febre Maculosa das Montanhas Rochosas/epidemiologia , Febre Maculosa das Montanhas Rochosas/imunologia , VirginiaRESUMO
Protective immunity to Rickettsia tsutsugamushi in a murine model is dependent upon the development of cell mediated immunity, as demonstrated by lymphocyte transfer, production of lymphokines and -interferon by thymus-derived lymphocytes, activation of macrophages by lymphokines and demonstration of delayed-type hypersensitivity response following exposure to these organisms. Infection of mice with small numbers of Rickettsia typhi inoculated by a peripheral route leads to a more complex pattern of immune development, with a distinction between resistance to local and systemic infection. Nevertheless, thymus-derived lymphocytes and activated macrophages play a major role in modulating pathogenesis of infection, and delayed-type hypersensitivity responses are evident. Spotted fever group rickettsiae also elicit a cell-mediated response in rodents, and protection against Rickettsia conorii infection has been achieved by adoptive transfer of thymus-derived lymphocytes from immune animals. Limited studies with Rickettsia akari suggest that activation of mouse macrophages is critical to host survival following infection with this organism.
Assuntos
Infecções por Rickettsia/imunologia , Animais , Hipersensibilidade Tardia , Imunidade Celular , Imunização , Interferons/biossíntese , Linfócitos/imunologia , Linfocinas/fisiologia , Ativação de Macrófagos , Camundongos , Tifo por Ácaros/imunologiaRESUMO
Eight sublines of C3H mice were tested for their resistance to lethal infection with Rickettsia akari,. strain Kaplan. C3H/HeJ mice were unique in their susceptibility to approximately one plaque-forming unit of rickettsiae. This lack of resistance is apparently due to a mutation in the mouse strain which occurred after 1950.
Assuntos
Imunidade Inata , Mutação , Infecções por Rickettsia/imunologia , Animais , Camundongos , Camundongos Endogâmicos C3H , Infecções por Rickettsia/genéticaRESUMO
The temperature range for optimum growth of Rickettsia conorii in suspension culture of gamma-irradiated L cells was 32-38 degrees C, resulting in rickettsial doubling times between 4.1 and 6.0 hr. An asynchronous release of R. conorii from host cells was suggested by the constant increase in percent cells infected over a 36 hr period. Rickettsial growth was optimal at neutral to slightly alkaline extracellular pH levels. A moderately acidic pH, however, resulted in an increase in doubling time from 4.1 to 7.8 hr.
Assuntos
Células L/efeitos da radiação , Rickettsia/crescimento & desenvolvimento , Temperatura , Animais , Divisão Celular , Raios gama , Concentração de Íons de Hidrogênio , CamundongosAssuntos
Genética Médica , Erros Inatos do Metabolismo/terapia , Polyomavirus/crescimento & desenvolvimento , Animais , Linhagem Celular/metabolismo , Centrifugação com Gradiente de Concentração , DNA Bacteriano , Desoxirribonucleases , Embrião de Mamíferos , Camundongos , Modelos Biológicos , Biologia Molecular , Hibridização de Ácido Nucleico , Timidina/metabolismo , Trítio , Proteínas Virais , Replicação ViralAssuntos
Imunidade Celular , Orientia tsutsugamushi/imunologia , Tifo por Ácaros/imunologia , Doença Aguda , Animais , Doença Crônica , Modelos Animais de Doenças , Hipersensibilidade Tardia , Imunização Passiva , Camundongos , Camundongos Endogâmicos , Tifo por Ácaros/microbiologia , Especificidade da EspécieRESUMO
Mechanisms of innate resistance to infection with the Gilliam strain of Rickettsia tsutsugamushi were examined using congenic strains of mice resistant (C3H/RV) or susceptible (C3H/He) to intraperitoneal infection. Both strains of mice were resistant to infection with 1,000 50% mouse lethal doses of rickettsiae if given intravenously. In both systems rickettsial replication occurred after intravenous infection, as evidenced by an increase in rickettsial numbers in the spleens of infected animals, followed by a decrease in rickettsiae to low levels by day 14 postinfection. Administration of the antimacrophage agents silica and carrageenan to C3H/He mice intravenously rendered these animals susceptible to lethal infection. Neither irradiation nor silica given individually rendered C3H/RV mice susceptible to intravenous infection. However, if silica and irradiation were given together, a lethal infection occurred after intravenous infection. C3H/RV mice became susceptible to lethal infection after sublethal doses of irradiation only if they were infected intraperitoneally. Administration of silica or carrageenan had no effect on the outcome of intraperitoneal infection of these mice with Gilliam rickettsiae. These data suggest that both strains of mice share innate resistance mechanisms to intravenous infection that consist of fixed macrophages. Resistance of C3H/RV mice to intraperitoneal infection, in contrast, apparently was dependent only on an irradiation-sensitive process.
Assuntos
Macrófagos/imunologia , Tifo por Ácaros/imunologia , Animais , Carragenina/farmacologia , Feminino , Raios gama , Imunidade Ativa , Imunidade Inata , Contagem de Leucócitos , Camundongos , Camundongos Endogâmicos C3H , Monócitos , Orientia tsutsugamushi/crescimento & desenvolvimento , Tifo por Ácaros/microbiologia , Dióxido de Silício/farmacologia , Baço/microbiologiaRESUMO
Two strains of C3H mice differed in their susceptibility to lethal infection with Rickettsia tsutsugamushi strain Gilliam. Adult C3H/RV mice were markedly more resistant to lethal infection than C3H/HeDub mice, and both were histocompatible as assessed by mixed-lymphocyte cultures and graft-versus-host responses. The inflammatory response of susceptible C3H/HeDub mice to intraperitoneal infection was evident approximately 5 days postinfection, and the magnitude of the cellular influx increased until death of the animal. The inflammation consisted of an early polymorphonuclear leukocyte response, followed by a mononuclear cell influx which persisted until death of the animal. The C3H/RV mice evidenced similar kinetics of cell influx, but the inflammatory response was significantly reduced in magnitude, and the response of C3H/RV animals to Gilliam was predominantly mononuclear in nature, with little influx of polymorphonuclear leukocytes into the peritoneal cavity. C3H/RV mice were rendered susceptible to Gilliam infection by induction of a nonspecific inflammation with thioglycolate if given 3 days after infection. Conversely, treatment of C3H/HeDub mice with indomethacin, an anti-inflammatory agent, prolonged survival after infection with Gilliam. The results of this study indicate that genetic resistance to Gilliam is not due simply to a greater host response to infection or, conversely, that susceptibility is due to a host response quantitatively lacking in a cellular component necessary for antirickettsial immunity.
Assuntos
Imunidade Inata , Camundongos Endogâmicos C3H/imunologia , Tifo por Ácaros/imunologia , Animais , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Indometacina/farmacologia , Inflamação , Camundongos , Camundongos Endogâmicos C3H/genética , Orientia tsutsugamushi/imunologia , Tifo por Ácaros/genética , Tioglicolatos/farmacologiaRESUMO
The development of antigen-responsive lymphocytes was followed in mice immunized with the Gilliam, Karp, or Kato strains of Rickettsia tsutsugamushi by utilizing an in vitro lymphocyte proliferation assay. Subcutaneous immunization with viable rickettsiae of all three strains resulted in the appearance of lymphocytes in the spleen responding to irradiated tissue culture-grown rickettsiae used as stimulating antigens. Although all animals demonstrated antigen-induced proliferation elicited by homologous antigen by 14 days after immunization, the time of peak responsiveness varied, depending on the strain of rickettsiae used for immunization. In all cases, peak proliferative responses occurred at a time after immunization that was after the previously reported time after immunization at which resistance to rechallenge was observed. Reactivity to heterologous strains of R tsutsugamushi developed roughly in parallel with homologous reactivity in Karp- and Gilliam-immunized mice, with a marked degree of heterologous reactivity evident. Kato-immunized mice demonstrated greater reactivity to heterologous antigens early in the development of antigen reactivity and demonstrated a somewhat greater degree of cross-reactivity, relative to homologous responses, than the other groups. It was found that nylon wool-nonadherent immune cells, if cultured with antigen and adherent cells obtained from normal spleens or peritoneal exudates, responded in culture. The thymus-derived lymphocyte nature of the responding cell was further suggested when treatment of immune spleen cells with anti-Thy 1.2 serum and complement eliminated antigen response.
Assuntos
Ativação Linfocitária , Tifo por Ácaros/imunologia , Animais , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/classificação , Antígenos de Bactérias/imunologia , Doença Crônica , Reações Cruzadas , Epitopos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Orientia tsutsugamushi/imunologia , Linfócitos T/imunologiaRESUMO
Antigens of plaque-purified Rickettsia tsutsugamushi strains Gilliam, Karp, and Kato were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were analyzed by an enzyme-linked immunosorbent assay. Six antigens were identified in each of the three prototype strains; in strain Gilliam, these antigens were located in the cell envelope fraction of the organisms. Reactivity of these isolated antigens with homologous or heterologous immune sera indicated that different macromolecules existed in all three strains, although they exhibited very similar mobilities during electrophoresis. Antigens of strain Gilliam reacted equally well with antibodies directed against Gilliam, Karp, or Kato rickettsiae. However, strains Karp and Kato each had two distinct antigens which did not react with heterologous antisera. R. tsutsugamushi antigens retained immunogenicity after electrophoresis, and antisera raised against them reacted with intact organisms and exhibited specificity in reactions with isolated antigens.
Assuntos
Antígenos de Bactérias/imunologia , Orientia tsutsugamushi/imunologia , Anticorpos Antibacterianos/biossíntese , Antígenos de Bactérias/análise , Antígenos de Bactérias/isolamento & purificação , Membrana Celular/imunologia , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Epitopos , Especificidade da EspécieRESUMO
The genetic basis for natural resistance to lethal infection with Rickettsia akari was studied in over 25 inbred strains, inbred hybrids, and outbred stocks of mice. Inbred mice infected intraperitoneally with the Kaplan strain of R. akari demonstrated three levels of response, susceptible (C3H/HeJ), intermediate (A/HeJ, A/J, A/WySn, BALB/cDub, BALB/cJ, and SJL/J), and resistant (AKR/J, AL/N, BALB/cAnN, BALB/cNCr1BR, C3H/HeN, C57BL/6J, C57L/J, CBA/J, DBA/2J, and SWR/J). No correlation was evident between the six H-2 haplo-types tested and susceptibility to Kaplan infection. Four outbred mouse stocks, Dub: (ICR), Wrc:(ICR), Caw:(CF1), and Mai:(S) were all resistant. The F1 inbred hybrids of resistant X resistant (AKD2F1/J), resistant X intermediate (CB6F1/U), intermediate X intermediate (CAF1/J), and resistant X susceptible (C3D2F1/J) parents were all resistant. The F2 and parental backcross generations of C3H/HeJ and DBA/2J hybrids yielded ratios of resistant to susceptible mice that suggested resistance was under multigeneic control. Susceptible mice (C3H/HeJ) were capable of mounting an immune response, since prior infection with the avirulent Hartford strain of R. akari rendered them resistant to subsequent lethal challenge with the Kaplan strains.
Assuntos
Imunidade Inata , Infecções por Rickettsiaceae/genética , Animais , Células Cultivadas , Cruzamentos Genéticos , Imunização , Camundongos , Camundongos Endogâmicos , Rickettsia/crescimento & desenvolvimento , Rickettsia/imunologia , Infecções por Rickettsiaceae/imunologiaRESUMO
Delayed-type hypersensitivity responses of inbred mice during the course of lethal and chronic infections with strains of Rickettsia tsutsugamushi were evaluated by using the influx of radiolabeled cells into antigen-injected ears. Congenic strains of C3H mice, which previously have been shown to be resistant (C3H/RV) or sensitive (C3H/HeDub) to lethal intraperitoneal infection with the Gilliam strain of rickettsiae, both expressed delayed-type hypersensitivity early in the course of infection (5 to 7 days). The sensitive C3H/HeDub mice, however, exhibited a marked decline in reactivity just before death. In contrast, reactivity of C3H/RV mice remained high through day 9 and declined slowly through day 15 after infection. Similar results were obtained when BALB/c mice were infected with either the Karp or the Gilliam strain of rickettsiae, which produce a lethal or nonlethal infection, respectively, in this strain of mice. Rechallenge of C3H/RV mice elicited a rapid increase in reactivity, suggesting a secondary memory response. To analyze delayed-type hypersensitivity during chronic infection, C3H/HeDub mice were immunized by subcutaneous infection with the Gilliam strain of R. tsutsugamushi, and both delayed-type hypersensitivity reactivity and resistance to intraperitoneal challenge were examined. Delayed-type hypersensitivity reactivity developed slowly and peaked at 21 days postimmunization, which correlated with resistance to intraperitoneal challenge. Delayed-type hypersensitivity reactivity declined thereafter, but resistance to intraperitoneal challenge remained through 28 days postimmunization. Delayed-type hypersensitivity reactivity increased after secondary challenge at 28 days, again suggesting antigen memory generated by primary immunization. Transfer of delayed-type hypersensitivity reactivity was accomplished by using immune thymus-derived splenic lymphocytes isolated with nylon-wool columns. Abrogation of the ability of immune spleen cells to transfer delayed-type hypersensitivity reactivity after treatment with anti-Thy 1.2 alloantiserum and complement further supported the view that delayed-type hypersensitivity responses to scrub typhus rickettsiae were mediated by thymus-derived lymphocytes.
Assuntos
Hipersensibilidade Tardia/imunologia , Tifo por Ácaros/imunologia , Animais , Feminino , Imunização , Cinética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Orientia tsutsugamushi/imunologia , Linfócitos T/imunologiaRESUMO
Six proteins, previously established as major constituents of intact organisms, were identified in cell envelopes obtained from intrinsically radiolabeled Rickettsia prowazekii. Extrinsic radioiodination of intact organisms conducted at 0.5 micronM iodide indicated that protein 4 was the most peripheral, although protein 1 also had reactive groups exposed on the surface of the organisms. A 10-fold increase in iodide concentration resulted in labeling of protein 2, and at 50 micronM iodide, all six major proteins were radiolabeled. Similar selective labeling was not achieved with R. conorii. Analysis of both typhus and spotted fever group organisms radiolabeled with galactose suggested that carbohydrate was associated with proteins 1, 3, and 4. Typhus soluble antigen included all major proteins except protein 2, which remained attached to particulate rickettsiae after ether extraction. Protein 4 appeared to be prominent in the surface topography of R. prowazekii, was a component of soluble antigen and may have an important role in rickettsiae-host interactions.
Assuntos
Antígenos de Bactérias/análise , Proteínas de Bactérias/análise , Rickettsia/análise , Antígenos de Superfície/análise , Carboidratos/análise , Glicoproteínas/análise , Proteínas de Membrana/análise , Rickettsia/imunologia , Rickettsia prowazekii/análise , Febre Maculosa das Montanhas Rochosas/microbiologiaRESUMO
Unirradiated Rickettsia tsutsugamushi and a component of gamma-irradiated Karp immunogen required for homologous immunity were more stable than the immunogen component that elicited heterologous (Kato strain) protection.
Assuntos
Antígenos de Bactérias , Orientia tsutsugamushi/imunologia , Animais , Congelamento , Raios gama , Camundongos , Orientia tsutsugamushi/patogenicidade , Orientia tsutsugamushi/efeitos da radiação , Tifo por Ácaros/imunologia , TemperaturaRESUMO
The development and duration of immunity to lethal scrub typhus infection was studied in BALB/c mice vaccinated with gamma-irradiated Rickettsia tsutsugamushi, strain Karp. One intraperitoneal injection containing approximately 10(8) 50% mouse lethal doses (MLD(50)) of irradiated organisms elicited an immune response protective against challenge with 10(5) MLD(50) of viable Karp. The same mass of immunogen given in three injections at 5-day intervals increased homologous (Karp strain) protection 25-fold and heterologous (Kato strain) protection 60-fold. Further temporal expansion of the immunization regimen did not increase protection. Subcutaneous vaccination provided significant, but lower, levels of protection than were achieved by intraperitoneal immunization, but the levels of cell-transferable immunity elicited by the two routes were approximately the same. Immunologically specific protection after intraperitoneal vaccination developed rapidly enough to provide resistance against simultaneous challenge with 200 MLD(50) of Karp. Homologous immunity was protective against a 10(6)-MLD(50) challenge 7 days after completion of the three-injection regimen, remained at that level for 3 months, dropped to 10(4) MLD(50) by 9 months, and was effective against a 50-MLD(50) Karp challenge at 12 months. Protection against heterologous challenge was first observed on day 17 and peaked on day 38, when the mice resisted a 10(5)-MLD(50) Kato challenge. Thereafter, heterologous protection waned rapidly and was not significant at 6 months.